JPH0532039B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a rapid, simple and accurate method for measuring the total amount of polyamines in a sample using an enzymatic method. Polyamines are non-protein, low molecular weight, aliphatic basic compounds that are widely distributed in the living world.
It is known as a substance that plays an important role in proliferation and the metabolism of nucleic acids, which forms the biochemical background thereof. Spermine, spermidine, putretsucine, and cadaverine are mainly present in mammalian bodies, and these are collectively referred to as total polyamines. In recent years, it has been reported that the total amount of polyamines in so-called body fluids such as urine, blood, and lymph of cancer patients increases significantly compared to normal people, and that the amount of total polyamines decreases with treatment. . Therefore, measurement of the total amount of polyamines is becoming an effective means in clinical examinations, such as in diagnosing cancer, determining the therapeutic effect of cancer, and diagnosing prognosis. Conventionally, the method for quantifying the total amount of polyamines has been by gas chromatography [Clin.Chem., Vol. 19, Nos. 904-907]
(1973)], method using an amino acid analyzer [FEBS Lett., Vol. 46, No. 305-
307 (1974)], High Performance Liquid Chromatography Method [J. Chromatography, Vol. 145, No. 141-146
(1978)] were mainly used. However, these chemical methods are difficult to apply to general clinical tests because they lack speed, require extremely complicated processing operations, cannot process many specimens, and require special equipment and facilities. It was hot. On the other hand, a method for measuring the total amount of polyamines using enzymes has recently been proposed (Japanese Patent Publication No. 56-36918, JP-A No. 59-2700, JP-A-58-141798 and JP-A-58
−146297). Among these methods, the method disclosed in Japanese Patent Publication No. 56-36918 is a method in which amine oxidase derived from germinated soybeans is applied to a sample containing free polyamine, and the generated hydrogen peroxide is colored and quantitatively determined by colorimetry. In the No. 1 method, samples containing free and conjugated polyamines are first treated with ascorbic acid oxidase to remove the effects of reaction inhibitors, and then amine oxidase derived from germinated soybeans is treated to color the generated hydrogen peroxide. This is a colorimetric method. However, it has been found that all of these enzymatic methods for measuring the total polyamine content have the following drawbacks. These enzyme methods use amine oxidase derived from germinated soybeans, which produces 2 moles of hydrogen peroxide from 1 mole of spermine when spermine is used as a substrate, but other polyamines such as spermidine, Putrescine and cadaverine produce 1 mole of hydrogen peroxide, so when determining the total amount of polyamines in the sample,
Although the total amount of hydrogen peroxide produced as a result of the enzymatic reaction is determined colorimetrically, both of these methods yield values for spermine that are higher than the true value, and as a result, these methods lack accuracy. I end up. In particular, when blood containing a high spermine content is used as a sample for clinical testing, these enzymatic methods for measuring total polyamines have large errors and cannot be used. Furthermore, amine oxidase derived from germinated soybeans is a vegetable enzyme and is difficult to mass produce compared to enzymes derived from microorganisms, and therefore poses a cost problem when used in large quantities in clinical tests. On the other hand, the method of JP-A No. 58-141798 is a polyamine analysis method that uses Micrococcus flavidus putrescine oxidase in a polyamine solution.
This putretsusine oxidase is an enzyme that mainly acts on putretsucine, cadaverine, and spermidine, and has the disadvantage that it does not act on spermine among all polyamines, so blood cannot be used as a sample. discloses a method for quantifying total polyamines consisting of spermine, spermidine, acetylspermine, and acetylspermidine in a sample using polyamine oxidase M produced by the genus Penicillium, but this polyamine oxidase M does not act on putretsucine and cadaverine. This method also cannot be used to accurately measure the total amount of polyamines because of the fact that 2 moles of hydrogen peroxide are produced from 1 mole of spermine by acting on spermine. In view of these circumstances, the present inventors have conducted extensive studies to develop a method for measuring the total amount of polyamines using an enzyme method that is faster, more accurate, and more precise than the conventional method for measuring the amount of total polyamines. . As a result, spermine in a sample containing various polyamines was first converted to spermidine and/or putretsucine by polyamine oxidase,
After erasing the hydrogen peroxide produced at that time, an amine oxidase that acts on spermidine, putretsucine, and cadaverine but does not produce putrescine from spermidine is added and reacted, and the hydrogen peroxide produced at that time develops a color. If the total amount of polyamines is determined by colorimetric determination, this method produces an equimolar amount of hydrogen peroxide with spermine in the sample, making it possible to quickly and accurately measure the total amount of polyamines. After realizing this, the present invention was completed. One of the characteristics of the method of the present invention is that a pretreatment reaction is performed when measuring the total amount of polyamines in a sample using an enzyme. That is, as a pretreatment reaction, polyamine oxidase is applied to a sample containing total polyamines in advance to convert spermine in the sample into equimolar amounts of spermidine and/or putretzine according to the formula.

[Table] ↓
H ₂ N(CH ₂ ) ₄ NH ₂ (putrescine)
The reaction products obtained during the above-mentioned pretreatment are not necessarily constant, but products in different states can be obtained depending on conditions such as temperature and pH. In other words, when spermine is completely decomposed to spermidine and to putretzine, when spermine is decomposed to spermidine and stopped there, and when spermine is decomposed to spermidine and a part of spermidine is decomposed to putretzine (in this case, spermidine and putretzine are ) and so on. In the present invention, any of these cases can be used in the subsequent second reaction, but when using a known putrescine oxidase, it is more preferable that spermine is completely converted to putrescine in consideration of reactivity. . Next, the second feature of the method of the present invention is that the hydrogen peroxide produced as a result of the above pretreatment reaction is eliminated. The reason for this is that if the hydrogen peroxide produced in the pretreatment step remains, it will affect the next reaction and cause measurement errors, so the hydrogen peroxide produced in this step must be completely removed.
For this purpose, general hydrogen peroxide removal methods include adding catalase to decompose hydrogen peroxide, or reacting with one of the chromogens that react with hydrogen peroxide in the presence of peroxidase. can be used. These pretreatment reactions convert spermine into equimolar amounts of spermidine and/or putretzine. This is followed by a quantitative method for total polyamines. i.e. spermidine,
By treating a polyamine mixed sample consisting of putretzine and cadaverine with an amine oxidase that has the ability to decompose spermidine, putretzine, and cadaverine and does not produce putretzine from spermidine, it is possible to obtain equimolar excess of each from spermidine, putretzine, and cadaverine. Hydrogen oxide is produced. Therefore, the total amount of polyamines is determined by colorimetrically quantifying the hydrogen peroxide. Examples of reaction formulas of amine oxidase used in these second reactions are shown below as formulas (a) to (c).

[Table] ↓
_H2N ( _CH2 ) _3CHO (4-aminobutyraldehyde)

[Table] ↓
_H2N ( _CH2 ) _4CHO (5-aminovaleraldehyde)
The amine oxidases used in the above reaction formulas (a), (b), and (c) are spermidine, putretsucine,
Any substance that acts on cadaverine to produce equimolar hydrogen peroxide can be used. Next, the present invention will be explained in more detail with reference to Reference Examples and Examples, and methods for measuring amine oxidase activity, peroxidase activity, and catalase activity used in the second reaction with polyamine oxidase will be described. The activity units of polyamine oxidase and amine oxidase are measured as follows, and
One unit is defined as the amount of enzyme required to produce 1 μmol of hydrogen peroxide. (1) Polyamine oxidase Dissolve 10 mg of 4-aminoantipyrine, 0.2 ml of phenol, and 5 mg of peroxidase (manufactured by Boehringer, grade) in 100 ml of 0.1M phosphate buffer (PH6.5) to prepare a coloring solution. 1.5ml of this coloring solution and 0.5ml of 10mM spermidine
After preheating the mixture at 35°C for 3 minutes, 0.5ml of the enzyme solution was added and reacted. Then, using 6250 as the coloring molecule extinction coefficient at 505 nm, the enzyme activity is determined from the amount of change in absorbance at 505 nm (ΔA for 1 minute from the start of the reaction) due to the generated hydrogen peroxide. (2) Amine oxidase used in the second reaction As a typical example, the measurement of the activity of putrescine oxidase is as follows. Enzyme activity was determined in exactly the same manner as in (1) except that 0.5 ml of 10 mM putretsucine was used as the substrate and a phosphate buffer of PH 8.5 was used as the coloring solution buffer. (3) Peroxidase activity uses pyrogallol and hydrogen peroxide as substrates in a reaction at PH6.0 and 20℃.
The amount of enzyme that produced 1 mg of purpurogalin in 20 seconds was defined as 1 unit. (4) Catalase activity uses hydrogen peroxide as a substrate, and PH
7.0, the amount of enzyme required to decompose 1 μmol of hydrogen peroxide per minute in a 25°C reaction was defined as 1 unit. Reference Example 1 7.5 mg of 4-aminoantipyrine and 350 units of peroxidase were dissolved in 100 ml of 0.2M phosphate buffer PH7.1 to prepare a coloring solution. (A) 100nmol of spermine solution in 1.35ml of coloring solution
(1.60ml) and preheated at 30℃ for 3 minutes, then
-Ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine sodium salt (hereinafter abbreviated as TOOS) 0.45 mg and polyamine oxidase AT-1 (described in JP-A-56-92788)
0.15 units (50μ) were added. After reacting for 10 minutes at 30°C, absorbance at 555 nm was measured (Absorbance A) (B) 100 nmol of spermine solution in 1.35 ml of coloring solution
(1.50ml) and preheated at 30℃ for 3 minutes,
Polyamine oxidase AT- containing TOOS 0.45mg
10.15 units (50μ) were added. After reacting at 30°C for 10 minutes, 0.65M NaOH solution (0.1ml) was added (at this pH, polyamine oxidase does not act and putrescine oxidase acts easily), and then putrescine oxidase [agri It was prepared according to Cultural Biological Chemistry (Agric.Biol.Chem.) Vol. 30, p. 1202 (1966). 〕4.5 units (10μ)
was added and reacted at 30°C for 10 minutes, and then the absorbance at 555 nm was measured. (Absorbance B) (C) 100 nmol of spermine solution in 1.35 ml of coloring solution
(1.20 ml) and 20 units of catalase (0.2 ml), preheated at 30℃ for 3 minutes, and then added polyamine oxidase.
0.15 units of AT-1 (10μ, not including TOOS) was added. After reaction at 30℃ for 10 minutes, 0.65M
0.1 ml of NaOH solution and 0.1 ml of 30 mM sodium azide
ml, and 4.5 units (50 μ) of putrescine oxidase containing TOOS were added. 30℃, 10
After a minute of reaction, absorbance at 555 nm was measured. (Absorbance C) The absorbance in the above reaction was as follows. Absorbance A = 1.109 (approximately equivalent to 200 nmol of hydrogen peroxide) Absorbance B = 1.664 (approximately equivalent to 300 nmol of hydrogen peroxide) Absorbance C = 0.552 (approximately equivalent to 100 nmol of hydrogen peroxide) Reaction C When TOOS (containing 0.45 mg) solution was added to the reaction solution with polyamine oxidase, the absorbance was 0.000. That is, absorbance A indicates that spermine was completely oxidized to putrescine in reaction A, and 2 molar amounts of hydrogen peroxide were produced. Absorbance B indicates that in reaction B, 2 moles of hydrogen peroxide and 1 mole of putretzine are produced by the reaction between spermine and polyamine oxidase, and then putrescine is completely oxidized by putrescine oxidase, resulting in a total of 3 Indicates that moles of hydrogen peroxide were produced. Absorbance C indicates that 2 molar amounts of hydrogen peroxide produced together with 1 molar amount of putretsucine in reaction C with polyamine oxidase are completely decomposed by catalase added at the same time (after this treatment). (It is necessary to inactivate the catalase activity by adding sodium azide.) Then, the produced putrescine was decomposed with putrescine oxidase to produce 1 mole of hydrogen peroxide. That is, in the method of reaction C, hydrogen peroxide is produced in an amount equal to the mole of spermine, and the number of moles of hydrogen peroxide is the number of moles of spermine in the sample. Reference example 2 Spermidine instead of 100nmol of spermine
The same study as in Reference Example 1 was conducted using 100 nmol, and the results were as follows. Absorbance A = 0.554 (equivalent to 100 nmol of hydrogen peroxide) Absorbance B = 1.112 (equivalent to 200 nmol of hydrogen peroxide) Absorbance C = 0.556 (equivalent to 100 nmol of hydrogen peroxide) Polyamine oxidase in reaction C In the reaction solution with
The absorbance when TOOS (containing 0.45 mg) solution was added was 0.000. That is, absorbance A indicates that spermidine is completely oxidized to 1 molar amount of putretsucine by polyamine oxidase in reaction A, and 1 molar amount of hydrogen peroxide is produced, and absorbance B indicates that spermidine is completely oxidized to polyamine oxidase in reaction B. As a result, it is oxidized to produce 1 molar amount of hydrogen peroxide and 1 molar amount of putrescine,
This shows that an additional 1 mole of hydrogen peroxide was produced from the putrescine, for a total of 2 moles of hydrogen peroxide. In addition, the absorbance C is determined by the reaction C in which 1 molar amount of hydrogen peroxide, which is produced together with 1 molar amount of putrescine by the reaction of spermidine with polyamine oxidase, is completely decomposed by catalase (after this, catalase reacts with sodium azide). (It was completely inactivated by the addition of 1 molar amount of hydrogen peroxide.) Then, by reacting the putrescine produced with putrescine oxidase, only 1 molar amount of hydrogen peroxide was measured. That is, by employing the method of Reaction C, an equimolar amount of hydrogen peroxide is produced from spermidine, and therefore, when hydrogen peroxide is quantitatively determined, it is immediately clear that it is the amount of spermidine in the sample. Reference example 3 Putrescine instead of 100nmol of spermine
The same study as in Reference Example 1 was conducted using 100 nmol or 100 nmol of cadaverine, and the results were as follows. Putretsucine Cadaverine Absorbance A 0.000 0.000 Absorbance B 0.554 0.555 Absorbance C 0.553 0.554 In other words, polyamine oxidase is putretsucine,
It was shown that cadaverine was not oxidized at all, but was degraded by putrescine oxidase, producing equimolar amounts of hydrogen peroxide. Reference Example 4 Add color developing solution and 100 nmol (1.50 ml) of each substrate (spermine, spermidine, putrescine, or cadaverine) to 1.35 ml of color developing solution and each substrate.
Add 0.65M NaOH solution (0.1ml) and incubate at 30℃ for 3
After prewarming for minutes, 4.5 units (50 μ) of putrescine oxidase containing 0.45 mg of TOOS were added. 30℃
After reacting for 10 minutes, the absorbance at 555 nm was measured and the results were as follows. Type of substrate Absorbance Spermine 0.000 Spermidine 0.555 Putretsucine 0.553 Cadaverine 0.556 In other words, it was shown that putretzine oxidase does not use spermine as a substrate at all, but oxidizes spermidine, putretsucine, and cadaverine, each producing equimolar amounts of hydrogen peroxide. . Reference Example 5 The same procedure as in Reference Example 1 was performed using 10 to 100 nmol of spermine or spermidine as a substrate. The results are shown in Table 1.

[Table] That is, in reaction C, it was shown that hydrogen peroxide in amounts corresponding to the amounts of spermine and spermidine could be measured in the range of 10 to 100 nmol each, and the total amount of polyamines could be measured very accurately. Reference example 6 10mM spermine, 10mM spermidine, 10m
Individual samples of M putrescine and 10mM cadaverine, each of these four polyamines were mixed in a ratio of 1:1:1:
Sample mixed at a ratio of 1 (mixed sample A) and 2:2:
Six types of samples mixed at a ratio of 1:1 (mixed sample B) were prepared as substrates. 1.35 ml of coloring solution, 1.20 ml of distilled water, and 20 units of catalase (0.2 ml) were added to each substrate (10 μ), and after preheating at 30° C. for 3 minutes, 0.15 units of polyamine oxidase (10 μ) was added. After reaction at 30℃ for 10 minutes, add 0.65M NaOH solution 0.1
ml and 0.1 ml of 30mM sodium azide were added, and 4.5 units (30μ) of putrescine oxidase containing 0.45mg of TOOS were added. After reaction at 30℃ for 10 minutes
The results of measuring the absorbance at 555 nm were as follows. Type of substrate Absorbance Spermine 0.555 Spermidine 0.553 Putrescine 0.554 Cadaverine 0.554 Mixed sample A 0.557 Mixed sample B 0.555 In other words, it was shown that the present method can be used with a single solution of each polyamine or any mixed solution. Reference Example 7 Coloring solutions A-d were prepared as follows, and 1.35 ml of each of A-D and substrate (10mM spermidine or
Mix 0.10ml of 1.0mM spermine), 0.10ml of 30mM sodium azide solution, and 1.25ml of distilled water, and heat at 30°C.
Preheated for 3 minutes. Add polyamine oxidase to this solution.
10.15 units (50μ) of AT was added and reacted at 30°C for 10 minutes. After the reaction is completed, add 0.01 ml of coloring solutions a to d to the reactions using coloring solutions A to D, respectively.
Add 0.10ml of 0.65M sodium hydroxide solution,
Absorbance at 555 nm was measured (absorbance A). Next, 4.5 units of putrescine oxidase (50μ
) and continue the reaction at 30℃ for 10 minutes.
The absorbance of the sample was measured (absorbance B). The results are shown in Table-2.

[Preparation of coloring solution]

Coloring solution A: 7.5mg of 4AA was added to 0.2M phosphate buffer (PH
7.1) Color developer B: 7.5 mg of 4AA and 350 units of peroxidase were dissolved in 100 mL of 0.2M phosphate buffer (PH7.1) Color developer C: 33.7 mg of TOOS was dissolved in 0.2M phosphate buffer (PH7.1).
Dissolve in 100ml of 7.1) Coloring solution D: TOOS 33.7mg and peroxidase 350
Dissolve the unit in 100ml of 0.2M phosphate buffer (PH7.1) Coloring solution a: TOOS 44.5mg and peroxidase 350
Dissolve the unit in 1.0ml of 10mM phosphate buffer (PH7.0) Coloring solution B: Dissolve 44.5mg of TOOS in 1.0ml of 10mM phosphate buffer (PH7.0) Coloring solution C: 10.0mg of 4AA and peroxidase 350 Dissolve the unit in 1.0ml of 10mM phosphate buffer (PH7.0) Color developer d: 10.0mg of 4AA in 10mM phosphate buffer (PH7.0)
Dissolved in 1.0ml of 7.0) As is clear from Table 2, B and b, that is, B-
It was found that the amount of spermine and spermidine in the reaction solution could be accurately measured by the combination of b, D and d, that is, D-d. That is, the hydrogen peroxide produced when spermidine and spermine are oxidized with polyamine oxidase is eliminated by reacting with only one of the chromogens used for measuring hydrogen peroxide in the presence of peroxidase, and the color is then removed. The total amount of polyamines could be accurately measured by allowing putrescine oxidase to act in a mixed system of the drug substance. Reference example 8 TOOS 33.7 mg, peroxidase 350 units
Dissolved in 100ml of 0.2M phosphate buffer (PH7.1),
A coloring solution was prepared. 10 to 100 nmol of spermine, 10 to 100 nmol of spermidine to 1.35 ml of this coloring solution,
Putretsucine 10~100nmol, cadaverine 10~
Distilled water (liquid volume: 2.70 ml) was added to 100 nmol and each of Sample A (10 μ) and Sample B (10 μ) prepared in the same manner as in Reference Example 6, and preheated at 30° C. for 3 minutes. Next, 0.15 units (50μ) of polyamine oxidase AT-1 was added and reacted at 30°C for 10 minutes. Add 4-aminoantipyrine solution (1.0
mg/ml) 0.1ml and 0.65M sodium hydroxide solution.
0.1 ml was added and the absorbance at 555 nm was measured (absorbance A). Furthermore, putrescine oxidase was added to this reaction solution.
After adding 4.5 units (50 μ) and continuing the reaction at 30° C. for 10 minutes, absorbance at 555 nm was measured. The results are shown in Table-3 and Table-4.

【table】

[Table] In other words, hydrogen peroxide generated when polyamine oxidase AT-1 oxidizes spermine or spermidine is removed by the reaction in the presence of TOOS-peroxidase, and the value obtained for absorbance B is It reflected the concentration of the substrate. Therefore, the total amount of sample A and sample B was also measured, and the hydrogen peroxide produced in the first stage reaction (reaction with polyamine oxidase) was removed.
It was shown that the total amount of polyamines can be easily measured by applying putrescine oxidase. In this way, as is clear from Reference Examples 1 to 8, samples containing various polyamines are first treated with polyamine oxidase, and the hydrogen peroxide produced at that time is used as a chromogen that reacts with catalase or peroxidase and hydrogen peroxide. The total amount of polyamines in a sample can be easily and accurately measured by eliminating the polyamines by adding acetate, and then applying an amine oxidase that acts on spermidine, putretsucine, and cadaverine, but does not produce putretsucine from spermidine. I realized that. Example 1 25μ or 50μ of a solution prepared by mixing 10mM solutions of spermine, spermidine, putretsucine, and cadaverine in a ratio of 2:2:1:1 was added to 5.0ml of blood, and after stirring vigorously for 3 minutes,
The supernatant was collected by centrifugation at 3000 rpm for 5 minutes (6.2 ml). Neutralize this supernatant (pH around 6-7)
It was adsorbed onto an Amberlite CG-50 column (0.5 x 1 cm). After washing the column with distilled water (3.0ml),
Elute polyamines with 0.5M hydrochloric acid (3.0ml),
0.67M sodium hydroxide solution (2.0ml) was added to neutralize. This neutralized solution was used as a sample for the following reaction. Add 1.50ml of the above neutralizing solution and catalase to 1.35ml of coloring solution.
Add 50 units (25μ) and preheat at 30℃ for 3 minutes, then polyamine oxidase AT-1 0.15 units (25μ)
was added and further heated for 10 minutes. Next, 4.5 units (50 μ) of putrescine oxidase containing 1.35 M sodium hydroxide solution (50 μ), 60 mM sodium azide, and 0.45 mg TOOS were added, and the mixture was further reacted at 30° C. for 10 minutes. The absorbance of the reaction solution at 555 nm was as follows. No addition 0.053 Addition of 25μ 0.312 Addition of 50μ 0.575 When the addition recovery rate was calculated from these values, it was very good at 100.4% when adding 25μ and 101.1% when adding 50μ. Example 2 5 ml of 12N hydrochloric acid was added to 20 ml of urine, and the mixture was hydrolyzed at 100° C. for 3 hours to convert the bound polyamine into free polyamine. (The precipitate generated during hydrolysis was removed by centrifugation (5000 rpm, 5 minutes), and the supernatant fraction was adjusted to pH around 6 with 10N sodium hydroxide solution, and distilled water was added to make 60 ml.) The neutralized solution was adsorbed onto an Amberlite CG-50 column (1 x 2.5 cm), and the column was filled with 20 ml of distilled water and 0.5 N hydrochloric acid solution.
Washed with ml. Next, from this column, 0.5N hydrochloric acid 10
The polyamine was eluted with 10 N sodium hydroxide solution, the pH was adjusted to around 7.5, and the volume was made up to 20 ml with distilled water.
As a result of performing the same reaction as in Example 1 using this neutralized solution as a sample, the absorbance at 555 nm was 0.445.
It was hot. This value indicates that 80.1 nmol of polyamine was present in the reaction solution. In other words, it was shown that 1 ml of urine contained 53.4 nmol of polyamine.
In addition, we concentrated the neutralized solution 5 times and measured polyamines using an amino acid analyzer using 100μ of the solution (the measurement method was as described in [Agricultural Biological Chemistry (Agric.Biol.Chem.)]. 44 volumes,
2467-2475 (1980)]. ) It was shown that 1 ml of urine contained 52.7 nmol of polyamines, indicating that this method correlated very well with chemical analysis methods.

Claims (1)

[Claims] 1. After treating various polyamine-containing samples with polyamine oxidase to convert spermine in the sample to spermidine and/or putretzine and erasing the hydrogen peroxide generated at the time, the enzyme further acts on spermidine, putretzine, and cadaverine, and A method for measuring the total amount of polyamines, which comprises adding an amine oxidase that does not produce putrescine from spermidine, causing a reaction, and determining the amount of polyamines in a sample by quantifying the amount of hydrogen peroxide produced.